Recent advances in monoclonal antibody therapy of cancer indicate that this therapeutic strategy is beginning to realize its potential after twenty years of intensive preclinical and clinical trial research. The central hypothesis of our work continues to be that multifunctional antibody-based proteins targeting tumors and host effector cells can stimulate host-protective anti-tumor immune responses. This hypothesis has been addressed through the construction and testing of bispecific antibodies (BsAb) that efficiently promote targeted cellular cytotoxicity. In the current funding period, we have shown that whole IgG bispecific antibodies targeting tumor antigens and human effector cell trigger molecules cause systemic toxicities that restrict antibody doses and efficacy. The potential of targeted cellular therapy is also restricted by the limited availability of potentially inflammatory effector cells in the tumor microenvironment. Using human phage- displayed single-chain Fv fragment-based BsAb as templates, we will systematically modify these structures to develop improved reagents and strategies to promote targeted tumor inflammation. The first specific aim is to optimize the structure of bispecific antibodies (BsAb) targeting HER2/neu and human Fc-gamma-RIII for tumor targeting and cytotoxicity promotion. We hypothesize that increasing the size and span of this BsAb will enhance its tumor- specific cytotoxicity properties. C6B1D2-Y3, a single-chain Fv (scFv) dimeric BsAb with high affinity for HER2/neu and lower affinity for human Fc-gamma-RIII will serve as the template for these efforts. The second specific aim is to introduce leukocyte chemotactic or proliferative moieties into a structurally optimized antibody or BsAb format to promote leukocyte infiltration into tumor sites. We hypothesize that this will enhance the accumulation of potentially cytolytic effector cells at tumor sites, facilitating BsAb-directed therapy. The third specific aim is to develop antibody-targeting strategies to assemble the bacterial superantigens, staphylococcal enterotoxin A or B, specifically on tumor cells to promote in situ antibody-targeted superantigen- mediated cytotoxicity. We will test the hypothesis that antibody- directed assembly of intact superantigens at tumor sites from biologically inactive fragments will facilitate tumor-specific T cell activation. If validated, this will lead to clinical trials of antibody-directed superantigen assembly on tumor cell surfaces and will demonstrate the feasibility of this general strategy for tumor-specific immunomodulation. The successful achievement of these aims will identify new therapeutic reagents and strategies.